An in vitro system for the rapid functional characterization of genes involved in carotenoid biosynthesis and accumulation
An in vitro system for the rapid functional characterization of genes involved in carotenoid biosynthesis and accumulation
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DOI：
10.1111/tpj.12384
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发表时间：
2014-02-01
期刊：
影响因子：
7.2
通讯作者：
Zhu, Changfu
中科院分区：
文献类型：
--
作者：
Bai, Chao;Rivera, Sol M.;Zhu, Changfu
我们建立了一种基于水稻胚性愈伤组织的快速代谢基因功能鉴定方法。我们验证了使用选择的良好表征的基因在类胡萝卜素生物合成途径中的已知功能的测定，允许基于组织颜色的愈伤组织表型的快速视觉筛选。然后，我们使用该系统来确定两个未表征的基因的功能：化学合成的β-胡萝卜素酮醇酶基因优化玉米密码子的使用，和野生型拟南芥的花椰菜橙子基因的直系同源物。与以前的报告（洛佩斯，A.B.，货车Eck，J.，康林，B. J.，保罗，DJ，奥尼尔和李，L。（）J. Exp.机器人59，213-223; Lu，S.，货车Eck，J.，Zhou，X.，中国科学院，洛佩斯，A.B.，O'Halloran，D. M.，Cosman，K.M.，康林，B. J.，保罗，DJ，Garvin，D.F.，Vrebalov，J.，科钦，L. V.，Kupper，H.，厄尔，E. D.，Cao，J.和Li，L.（）Plant Cell 18，3594-3605），我们发现野生型橙子等位基因足以诱导质体分化。我们还发现，即使在没有橙子的情况下，通过增加前体的可用性，从而通过该途径驱动通量，也可以诱导质体分化。值得注意的是，我们发现不同的胚乳特异性启动子在水稻愈伤组织中高度活跃，尽管它们在成熟植株中的活性有限。我们的愈伤组织系统提供了一个独特的机会来预测代谢工程在复杂途径中的作用，并提供了一个起点，定量建模和合理设计的工程策略，使用合成生物学。我们讨论了我们的数据分析和工程类胡萝卜素生物合成途径的影响。
We have developed an assay based on rice embryogenic callus for rapid functional characterization of metabolic genes. We validated the assay using a selection of well-characterized genes with known functions in the carotenoid biosynthesis pathway, allowing rapid visual screening of callus phenotypes based on tissue color. We then used the system to identify the functions of two uncharacterized genes: a chemically synthesized -carotene ketolase gene optimized for maize codon usage, and a wild-type Arabidopsis thaliana ortholog of the cauliflower Orange gene. In contrast to previous reports (Lopez, A.B., Van Eck, J., Conlin, B.J., Paolillo, D.J., O'Neill, J. and Li, L. () J. Exp. Bot. 59, 213-223; Lu, S., Van Eck, J., Zhou, X., Lopez, A.B., O'Halloran, D.M., Cosman, K.M., Conlin, B.J., Paolillo, D.J., Garvin, D.F., Vrebalov, J., Kochian, L.V., Kupper, H., Earle, E.D., Cao, J. and Li, L. () Plant Cell 18, 3594-3605), we found that the wild-type Orange allele was sufficient to induce chromoplast differentiation. We also found that chromoplast differentiation was induced by increasing the availability of precursors and thus driving flux through the pathway, even in the absence of Orange. Remarkably, we found that diverse endosperm-specific promoters were highly active in rice callus despite their restricted activity in mature plants. Our callus system provides a unique opportunity to predict the effect of metabolic engineering in complex pathways, and provides a starting point for quantitative modeling and the rational design of engineering strategies using synthetic biology. We discuss the impact of our data on analysis and engineering of the carotenoid biosynthesis pathway.